For many species of virus, the first virion to infect a cell will exclude any subsequent virion entry, preventing that cell from becoming ?superinfected?. This process, called superinfection exclusion (SIE) is important be- cause many aspects of viral pathology and transmission are affected by the number of virions that infect a single cell. In this proposal, we aim to discover the mechanism of an early form of Herpes simplex virus 1 (HSV-1) SIE and the transcriptional responses that govern exclusion in the nervous system. Currently, there is a critical knowledge gap regarding the mechanism and effectors of early exclusion preventing experimental manipulation of SIE. The ability to specifically target and manipulate SIE is necessary to study how SIE affects neuronal pathogenesis and spread in vitro and in vivo. The experiments in this proposal aim to bridge this gap by identifying how early HSV-1 SIE blocks infection, and the specific cellular and viral genes that contribute to early SIE. Our first aim will employ quantitative assays to identify the effect of SIE on secondary HSV-1 virion fusion and entry. Knowing the stage of entry that is blocked by SIE is a critical step in determining the overall mechanism. The goal of our second aim is to modulate the expression of individual viral and cellular genes to alter SIE phenotypes in vitro. Based on published literature and our own preliminary data, we have compiled a list of candidate genes known to be differentially expressed during HSV-1 infections. Using an ICP4-null strain of HSV-1 that does not support SIE, we have refined our list of genes to those that are differentially expressed between HSV-1 infection conditions that either do or do not induce SIE. Restoring the expression of individual genes under ICP4-null conditions to see if an SIE phenotype can be restored, and vice versa, will allow us to identify critical SIE effectors. This proposal will identify the mechanism of early HSV-1 SIE and produce the tools necessary to alter levels of exclusion through the manipulation of gene expression. Once these tools and targets have been obtained, we can quickly move into applicable in vitro and in vivo studies that will evaluate the effect of early HSV-1 SIE on neuronal transmission and viral pathogenesis. Understanding how HSV-1 induces early SIE will help us to understand the transmission of herpesviruses within the nervous system and potentially be exploited by new antiviral therapies directed at treating and preventing HSV-1 disease.